Interactions of proteins in the Bluetongue virus core

蓝舌病毒核心蛋白质的相互作用

• 批准号: 7676771
• 负责人: POLLY ROY
• 金额: $ 28.62万
• 依托单位: LONDON SCH/HYGIENE & TROPICAL MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676771
• 关键词: Affect; Animal Viruses; Animals; Architecture; Area; Biochemical; Biochemical Reaction; Biological Assay; Biology; Bluetongue virus; Capsid; Cells; Complex; Core Protein; Crystallization; Dissection; Enzyme Kinetics; Enzymes; Family member; Genetic; Genetic Transcription; Genome; Goals; High Pressure Liquid Chromatography; Human; In Vitro; Insect Proteins; Kinetics; Lead; Mammalian Orthoreovirus; Minor; Molecular; Molecular Machines; Mutagenesis; Nucleosome Core Particle; Orbivirus; Play; Polymerase; Proteins; Public Health; RNA-Directed RNA Polymerase; Reaction; Reagent; Recombinant Proteins; Recombinants; Reoviridae; Reoviridae Infections; Reovirus; Research; Resolution; Rest; Roentgen Rays; Role; Spectrum Analysis; Structural Protein; Structure; Structure-Activity Relationship; Surface Plasmon Resonance; System; Tertiary Protein Structure; Transcript; Transcriptase; Viral; Viral Genome; Viral Proteins; Virus; Virus Replication; Viviparous-1 protein; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; design; enzyme activity; helicase; in vitro Assay; in vitro activity; inhibitor/antagonist; light scattering; man; member; nanomachine; positional cloning; programs; protein expression; protein protein interaction; protein structure; reconstitution; rice dwarf virus; single molecule; three-dimensional modeling; virus core

DESCRIPTION (provided by applicant): Upon cell infection, Reoviridae capsids never completely disassemble, and viral transcripts are extruded from the icosahedral core particle. As a consequence the core must contain all the necessary enzymatic reactions for transcription and capping. The interactions between viral proteins and between these proteins and the viral genome in the core, is highly specific and the core may be considered to function as a precise molecular machine. Atomic structures for the cores of three members of this family (Bluetongue virus, Rice dwarf virus and mammalian orthoreovirus) are available, and are among the largest structures solved by X- ray diffraction. However, the core architecture of BTV (and RDV) is different with only reovirus having defined capping turrets. For BTV, capping protein is co-localised with the RNA-dependent-RNA-polymerase (RdRp) at the fivefold vertices of the core. Although the transcription complex within the core has been localised, it was not possible to resolve its structural details from the crystal structure. The objective of this application is to provide a complete structural and biochemical understanding of how the BTV transcription complex functions. Completion of this goal will pave the way for the rational design of inhibitors to block orbivirus replication and provide lead compounds for related viruses of pathogenic significance. Furthermore, the work will contribute to the growing areas of understanding the structural basis of transcription and for understanding how complex machines can be constructed from modular protein-based units. BTV is uniquely placed to deliver this information. It is the only non-turreted member of the Reoviridae for which an atomic core structure and complete in vitro activity of purified recombinant enzymatic proteins is available. The proposed research will build on these reagents and on our recent crystal structure of the BTV capping enzyme to understand the molecular basis of enzyme function and how the enzymatic proteins of the core act in concert to achieve transcription. In Specific Aim 1 we will undertake a detailed program of mutagenesis, biochemical and biophysical studies to understand the structure-function relationships of the four enzymatic activities of the capping enzyme. Specific Aim 2 will focus on the protein-protein interactions between the capping and RdRp proteins, and between these proteins and the major structural proteins of the core. Specific Aim 3 will focus on structure-function studies of the core associated viral helicase protein to understand if this protein is functional in transcription or packaging of the viral genome. Finally, in Specific Aim 4 recombinant proteins will be used to reconstitute viral transcription complexes in vitro to understand how these proteins interact with and affect the activity of each other. In all approaches we will use a combination of structural (NMR, cryo-EM, X-ray crystallography, dynamic and static light scattering) and biochemical (single molecule kinetic studies, mutagenesis, Raman and IR spectroscopy, HPLC, enzyme assays to follow kinetics, surface plasmon resonance) approaches. PUBLIC HEALTH RELAVANCE: The project aims to provide a complete understanding of how a complex virus functions as a nanomachine. Completion of this goal will pave the way for the rational design of inhibitors to block virus (in particular, an animal virus) replication and provide lead compounds for related viruses of pathogenic significance to humans and animals.

描述（由申请人提供）：在细胞感染后，旋圆叶胶状衣壳永远不会完全拆卸，病毒转录本被挤出于二十面体核心颗粒。结果，核心必须包含用于转录和封盖的所有必要的酶促反应。病毒蛋白之间，这些蛋白质与核心中病毒基因组之间的相互作用高度特异，核心可以被认为是精确的分子机。该家族三个成员的核心（Bluetongue病毒，水稻矮人病毒和哺乳动物矫形病毒）的原子结构可用，并且是X-Ray衍射解决的最大结构之一。但是，BTV（和RDV）的核心架构仅有不同的依孢病毒，只有定义的上限炮塔。对于BTV，上限蛋白与核心五倍顶点处的RNA依赖性RNA-聚合酶（RDRP）共定位。尽管核心内的转录复合物已定位，但不可能从晶体结构中解析其结构细节。该应用的目的是对BTV转录复合物如何功能提供完整的结构和生化理解。该目标的完成将为抑制剂的合理设计铺平道路，以阻止眼病毒复制，并为致病意义的相关病毒提供铅化合物。此外，这项工作将有助于理解转录的结构基础的不断增长领域，并了解如何从模块化蛋白质单元构建复杂机器。 BTV是独特的，可以提供此信息。它是reoviridae的唯一非关闭构件，可以使用原子核心结构和纯化重组酶蛋白的完全体外活性。拟议的研究将建立在这些试剂和我们最近的BTV限制酶的晶体结构的基础上，以了解酶功能的分子基础以及核心的酶蛋白如何协同作用以实现转录。在特定目的1中，我们将进行详细的诱变，生化和生物物理研究，以了解上限酶的四种酶活性的结构 - 功能关系。特定的目标2将重点放在上限和RDRP蛋白之间的蛋白质 - 蛋白质相互作用上，以及这些蛋白质与核心的主要结构蛋白之间的蛋白质相互作用。特定目标3将重点关注核心相关病毒酶蛋白的结构功能研究，以了解该蛋白在病毒基因组的转录或包装中起作用。最后，在特定的目标中，4重组蛋白将用于体外重建病毒转录复合物，以了解这些蛋白质如何与彼此的活性相互作用并影响。在所有方法中，我们都将结合结构（NMR，冷冻EM，X射线晶体学，动态和静态光散射）和生化（单分子动力学研究，诱变，拉曼和IR光谱，HPLC，HPLC，酶测定，遵循动力学，表面plasmon soonance）的方法。公共卫生救助：该项目旨在完全了解复杂病毒作为纳米机器的功能。该目标的完成将为抑制剂的合理设计铺平道路，以阻止病毒（尤其是动物病毒）复制，并为对人类和动物具有致病意义的相关病毒提供铅化合物。